The presence of viral nucleic acids represents a danger signal for the immune system which initiates an anti-viral response to impede viral replication and eliminate the invading pathogen1. Interferon (IFN) response is a major component of the anti-viral response and comprises the production of type I IFNs, IFN-α and IFN-β. An anti-viral response also comprises the production of various other cytokines, such as IL-12, which promote innate and adaptive immunity1.
In order to detect foreign nucleic acids, immune cells are equipped with a set of pattern recognition receptors (PRR) which act at the frontline of the recognition process and which can be grouped into two major classes: the Toll-like receptors and the RNA helicases.
Members of the Toll-like receptor (TLR) family have been implicated in the detection of long dsRNA (TLR3)2, ssRNA (TLR7 and 8)3,4, short dsRNA (TLR7)5 and CpG DNA (TLR9)6. The TLRs reside primarily in the endosomal membranes of the immune cells7,8 and recognize viral nucleic acids that have been taken up by the immune cells into the endosomal compartments9.
RNA helicases, such as RIG-I, MDA-5 and LGP-2, have been implicated in the detection of viral RNA10,11. In contrast to the TLRs, the RNA helicases are cytosolic and are expressed in a wide spectrum of cell types, including immune cells and non-immune cells, such as fibroblasts and epithelial cells12. Therefore, not only immune cells, but also non-immune cells which express one or more of the RNA helicase(s), are capable of detecting and responding to viral RNA.
Both the TLR and the RNA helicase systems co-operate to optimally detect viral RNA.
Given the abundance of host RNA present in the cytoplasm, it is an intricate task to specifically and reliably detect virus-derived RNA. Maximal sensitivity together with a high degree of specificity for “non-self” are required. Two major mechanisms appear to be in place in vertebrate cells to distinguish “non-self” from “self” nucleic acids via a protein receptor-based recognition system: (1) the detection of a pathogen-specific compartmentalization, and (2) the detection of a pathogen-specific molecular signature or motif.
Endosomal RNA is recognized by the TLRs as “non-self”4. Notably, host RNA can acquire the ability to stimulate an IFN response via the activation of TLRs under certain pathological situations13.
Furthermore, there exist structural motifs or molecular signatures that allow the pattern recognition receptors (PRRs) to determine the origin of an RNA. For example, long dsRNA has been proposed to stimulate an IFN response via TLR32, RIG-I11 and MDA-514. The present inventors recently identified the 5′ triphosphate moiety of viral RNA transcripts as the ligand for RIG-I16,19. Even though nascent nuclear endogenous host RNA transcripts initially also contain a 5′ triphosphate, several nuclear post-transcriptional modifications, including 5′ capping, endonucleolytic cleavage, and base and backbone modifications, of the nascent RNA transcripts lead to mature cytoplasmic RNAs which are ignored by RIG-I. In addition, blunt-ended short dsRNA without any 5′ phosphate group16 or with 5′ monophosphate24 has also been postulated to stimulate RIG-I. A clearly defined molecular signature has not yet been reported for the ssRNA-sensing TLRs, TLR7 and TLR8. However, the fact that certain sequence motifs are better recognized than others by the TLRs suggests that the nucleotide composition of the RNA, on top of endosomal localization, may represent basis for discriminating between “self” and “non-self” by the TLRs3-5, 10, 11, 17, 18.
Despite its pivotal role in anti-viral defense, the mechanism of viral RNA recognition by RIG-I is not yet fully elucidated. Therefore, there is a need in the art to better understand the recognition of viral and/or other non-self RNA by RIG-I. Furthermore, given the efficacy of IFN-α in various clinical applications, there is a need in the art to provide alternative agents for inducing IFN-α production in vitro and in vivo.
It is therefore an object of the present invention to further identify the structural motifs or molecular signatures of an RNA molecule which are recognized by RIG-I. It is another object of the present invention to prepare RNA molecules which are capable of activating RIG-I and inducing an anti-viral, in particular, an IFN, response in cells expressing RIG-I. It is a further object of the present invention to use said RNA molecules for inducing an anti-viral, in particular, an IFN, response in vitro and in vivo. It is an ultimate object of the present invention to use said RNA molecules for preventing and/or treating diseases or conditions which would benefit from an anti-viral, in particular, an IFN, response, such as infections, tumors/cancers, and immune disorders.
Cellular transformation and progressive tumor growth result from an accumulation of genetic and epigenetic changes that alter normal cell proliferation and survival pathways38. Tumor pathogenesis is accompanied by a process called cancer immunoediting, a temporal transition from immune-mediated tumor elimination in early phases of tumor development to immune escape of established tumors. The interferons (IFNs) have emerged as central coordinators of these tumor-immune-system interactions38. Due to their plasticity tumors tend to evade single-targeted therapeutic approaches designed to control proliferation and survival of tumor cells40. Tumors even evade immunotherapies that are directed at multiple tumor antigens41.
It is therefore a further object of the present invention to use said RNA molecules for inducing apoptose, or both for inducing an anti-viral, in particular, an IFN, response in vitro and in vivo and for inducing apoptosis in the same molecule.
There remains a need in the art for combinatorial approaches that suppress tumor cell survival and at the same time increase immunogenicity of tumor cells in order to provide more effective tumor treatments42, 43.